Cerebrospinal fluid (spinal fluid) corresponds to extracellular fluid of the central nervous system (CNS) and is separated from blood components by the blood-brain barrier. The expression of central nervous system-derived proteins present mainly in this spinal fluid is likely to increase or decrease along with the onset of central nervous system disease. Accordingly, a central nervous system-derived protein whose expression level correlates with a particular central nervous system disease can serve as a promising diagnostic marker for the disease (Non Patent Literature 1).
Even the spinal fluid, however, is composed mainly of serum proteins (80% or more of its components) leaked into the brain fluid circulation system and actually contains central nervous system-derived proteins only in a trace amount (Non Patent Literature 1). In addition, heretofore known techniques are hardly capable of selectively isolating such central nervous system-derived proteins from the spinal fluid. Thus, the diagnostic marker cannot be searched for easily.
On the contrary, if central nervous system-derived proteins are leaked into blood, a low invasive and promising test method for central nervous system disease may be achieved by detecting the central nervous system-derived proteins in blood. Unfortunately, the amount of central nervous system-derived proteins in blood is much smaller than that of central nervous system-derived proteins in spinal fluid. Thus, the central nervous system-derived proteins in blood are more difficult to detect than those in spinal fluid.
Currently known diagnostic markers for central nervous system disease are, for example, trace substances such as tau protein or Aβ42 peptide, which is a causative factor of Alzheimer's disease (AD), and a cytokine for inflammatory disease (Non Patent Literature 2). Although the tau protein is an excellent diagnostic marker for AD, the expression of this protein means neuronal death. In this respect, the tau protein is insufficient for early diagnosis intended for the treatment of AD (avoidance of neuronal death). Also, the tau protein increases in other dementia types and thus, is not an AD-specific marker. Disadvantageously, the amount of the Aβ42 peptide varies only after progression of the disease. The cytokine permits sensitive assay but has the disadvantage of poor disease specificity.
In Patent Literature 1, the present inventor has found and disclosed a diagnostic drug for AD by focusing on the sugar chains of glycoproteins in serum. The serum glycoproteins, however, are mostly derived from the liver. In addition, their concentrations largely vary due to various diseases unrelated to AD. For example, so-called acute phase proteins such as C-reactive protein (CRP), mannose-binding protein, fibrinogen, haptoglobin, and α1-antitrypsin are known to largely vary in their amounts due to inflammation associated with mild infection, burn, and small scars, etc. Thus, the detection or enrichment of a central nervous system marker coexisting in a trace amount with serum proteins has encountered undesired technical difficulty.
In Patent Literature 2, the present inventors have hypothesized that the central nervous system contains a glycoprotein having a unique sugar chain structure, and screened for proteins differing in their sugar chain moieties between spinal fluid and serum using various antibodies. As a result, the present inventors have found that spinal fluid contains heretofore known transferrin-2 having α2,6 sialic acid (hereinafter, referred to as “Sia-α2,6-Gal” in the present specification, unless otherwise specified) at a non-reducing terminus, which is also found in large amounts in serum, as well as transferrin-1 having N-acetylglucosamine (hereinafter, referred to as “GlcNAc” in the present specification, unless otherwise specified) but no sialic acid residue at a non-reducing terminus. This transferrin-1 has been shown to be secreted from a spinal fluid-producing tissue choroid plexus. The present inventors have further revealed that this protein can serve as an index marker for idiopathic normal pressure hydrocephalus (iNPH), which is a spinal fluid metabolic disorder (Patent Literature 2). This result suggested that such a spinal fluid glycoprotein derived from the central nervous system has GlcNAc at a non-reducing terminus of the sugar chain but is free from Sia-α2,6-Gal.
PVL lectin is known as lectin strongly binding to non-reducing terminal GlcNAc. It has been the common general knowledge of the art that the PVL lectin hardly binds to non-reducing terminal Sia-α2,6-Gal (Non Patent Literature 3). Thus, those skilled in the art can predict that a spinal fluid glycoprotein having non-reducing terminal GlcNAc but no sialic acid residue, i.e., a spinal fluid glycoprotein derived from the central nervous system, can be enriched selectively by use of the PVL lectin. At the same time, those skilled in the art can predict that a terminal Sia-α2,6-Gal-containing glycoprotein, i.e., a serum glycoprotein can be removed selectively by use of the PVL lectin. Nonetheless, the present inventors have revealed that, unlike the common general knowledge of the art, in actuality, the PVL lectin also binds, albeit partially, to transferrin-2 having terminal Sia-α2,6-Gal. For example, as shown in FIG. 11, transferrin-2 (TF-2) is recovered in both a binding fraction BF (lane 4) and a nonbinding fraction NF (lane 5) of a PVL lectin column. This result indicates that a spinal fluid glycoprotein is difficult to selectively enrich using PVL lectin beads alone from a sample (body fluid such as spinal fluid or serum) containing a large amount of a glycoprotein having Sia-α2,6-Gal at a non-reducing terminus of the sugar chain, such as a spinal fluid sample. Meanwhile, transferrin-2 exhibits strong affinity for SSA lectin and is recovered in a binding fraction BF (lane 2) of a SSA lectin column. Alternatively, central nervous system-derived spinal fluid transferrin-1 (TF-1) having only GlcNAc at a non-reducing terminus specifically binds to PVL lectin but do not bind to SSA lectin (lanes 7 to 10).